This proposal is strongly responsive to the Challenge mechanism in the Broad Challenge Area of (14) "Stem cells", and within the Specific Challenge Topic of 14-HL-101 "Develop molecular signatures for heart, vascular, lung, blood diseases by profiling reprogrammed induced pluripotent stem cells derived from affected individuals of defined genotype." The advent of induced pluripotent stem cell (iPS cell) technology offers new and profound opportunities to study human disease and to develop cellular therapies targeted to a specific cell population. Our understanding of many genetic diseases is limited by our inability to isolate and study the precise cell populations that are defective in these diseases. There is emerging evidence that failure of tissue stem cells, or genetic alterations that affect tissue stem cells, underlies the pathophysiology of many common human diseases, including neurological disease, arthritis and cancer. One area in which tissue stem cells have been particularly implicated is in diseases of the hematopoietic system, including bone marrow failure. Dyskeratosis congenita (DC) is an inherited form of bone marrow failure, in which blood stem cell function is markedly impaired. Aplastic anemia in patients with DC is accompanied by defects in other tissues, including oral mucosa, nails and lung, among other tissues. DC is caused by mutations in telomerase genes or genes encoding telomere binding proteins. The accelerated telomere shortening that occurs in DC impairs tissue stem cell function, making DC a genetic syndrome in which broad stem cell dysfunction leads to multi-systemic defects. Although telomerase is assumed to be partially defective in DC, no stem cell population has been isolated from DC patients for direct analyses and the characterization of the biochemical and cellular defects in DC have been necessarily indirect. We plan to reprogram fibroblasts from DC patients to iPS cells, which will facilitate analysis of the DC defect in a self-renewing population of stem cells. We will use proteomic, biochemical and cell biological approaches to understand the specific causes of a human stem cell disease according to the following aims: (1) To generate iPS cells from DC fibroblasts and to study telomere maintenance and self-renewal in culture (2) To understand the nature of the defects in the telomerase complex through biochemical and proteomic studies in DC iPS cells and (3) Analysis of differentiation of DC iPS cells through embryoid body formation, teratoma development and directed differentiation to hematopoietic stem cells (HSCs). PUBLIC HEALTH RELEVANCE: The discovery of iPS cell technology promises to revolutionize our understanding of human disease and to allow the development of new cellular therapies for regenerative medicine applications. The ability to reprogram a patient's fibroblasts to iPS cells creates the opportunity to expand human cells with a specific genetic defect and to study that defect in a defined cell population, either to understand the basic biology of the disease or to study potential therapeutics. Furthermore, the genetic defects in iPS cells can be repaired and the iPS cells used as a source for cellular therapies after differentiation to specific cell lineages. In this proposal, we will generate iPS cells from patients with dyskeratosis congenita, a human stem cell disease caused by mutations in the telomerase enzyme. We will study self-renewal and differentiation, and employ proteomics to understand the specific nature of the defect in this disease. These experiments will greatly inform our understanding of the role of stem cell function in bone marrow failure and in human disease.